Recent developments in DNA manipulation allows for effective introduction of exogenous genes into the chromosome of a host cell (gene knock-in). This allows the insertion of a protein coding cDNA sequence at a particular locus in an organism's genome, for example, insertion of a mutation or exogenous gene at a particular locus on a chromosome. For example, a point mutation can be introduced into a target gene by knock-in to model human genetic disorders. In addition, exogenous genes such as reporter genes (EGFP, mRFP, mCherry, tdTomato etc.) can be introduced by homologous recombination into a particular locus of the target gene, and can be used to track expression of the target gene and study its expression profiles by expression of the reporter gene.
In many circumstances, gene knock-in involves homologous recombination mechanisms in an organism. Under natural circumstances, the probability of homologous recombination between an exogenous targeting vector and the genome of a cell is very low, about 1/105 to 1/106. Spontaneous gene targeting typically occurs at a very low frequency in mammalian cells with an efficiency of 1 in a million cells. The presence of a double-strand break is often recombinogenic and increases the homologous recombination frequency by several thousand folds. See Jasin, 1996, “Genetic manipulation of genomes with rare-cutting endonucleases,” Trends in genetics: TIG 12(6): 224-228. In plants, the generation of a double-strand break in DNA is known to increase the frequency of homologous recombination from a background level of about 10−3-10−4 by a factor of approximately 100-fold. See Hanin et al., 2001, “Gene targeting in Arabidopsis,” Plant J. 28:671-77. Generation of genetically modified mice via homologous recombination was made possible by the establishment of murine embryonic stem cell lines. For example, targeting vectors can be constructed using bacterial artificial chromosome (BAC), and introduced into murine embryonic stem cells via transfection (e.g., electroporation). Positive embryonic stem cell clones are selected and injected into mouse blastocysts microscopic cell mass, and then implanted into a surrogate mouse to produce genetically engineered chimeric mouse. However, methods for establishing embryonic stem cell lines from other species have not been as successful and widely used.
Recently developed techniques, including ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9, and other site-specific nuclease technologies, made it possible to create double-strand DNA breaks at desired locus sites. These controlled double-strand breaks can promote homologous recombination at such specific locus sites. This process relies on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to such sequences and induce a double-strand break in the nucleic acid molecule. The double-strand break is repaired either by an error-prone nonhomologous end-joining (NHEJ) or by homologous recombination (HR).
Homologous recombination that occurs during DNA repair tends to result in non-crossover products, in effect restoring the damaged DNA molecule as it existed before the double-strand break, or generating a recombinant molecule by incorporating sequence(s) of a template. The latter has been used in gene targeting, protein engineering, and gene therapy. If the template for homologous recombination is provided in trans (e.g., by introducing an exogenous template into a cell), the double-strand break in the cell can be repaired using the provided template. In gene targeting, the initial double-strand break increases the frequency of targeting by several orders of magnitude, compared to conventional homologous recombination-based gene targeting. In principle, this method could be used to insert any sequence at the site of repair so long as it is flanked by appropriate regions homologous to the sequences near the double-strand break. Although this method has had success in various species such as mice and rats, the efficiency and success rate of homologous recombination remain low, preventing the method from being widely used. For instance, the method remains costly and technically challenging for both scientific and commercial use.
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